ProgramDirector/PrincipalInvestigator(Last,First,Middle):Bartel,DavidP. PROJECT SUMMARY {See instructions): RNA molecules with structure-tdependent functions play important roles throughout molecular biology, and the broad, long-term objective of this grant is to understand these roles. The experiments of this proposal focus on functions involving double-stranded RNA (dsRNA), with an emphasis on the mechanism, biology, and evolution of RNA interference (RNAi), whereby dsRNA triggers the destruction of corresponding cellular mRNAs. Although RNAi has been lost in Saccharomyces cerevisiae, it is present in related budding yeasts, including Saccharomyces castellii. These species use noncanonical Dicer proteins to process long dsRNA into small interfering RNAs (siRNAs), which are loaded into the Argonaute protein to direct silencing. This proposal seeks to extend the first phase of an R37 award exploring the mechanism, biology, and evolution of RNAi in budding yeast. The first specific aim, which was to determine the consequences of losing or restoring RNAi in yeast, is now extended to determine the consequences of restoring RNAi in zebrafish. These experiments will explore how fish cope with the introduction of a new gene-regulatory pathway and might enable RNAi to become an effective research tool in fish. The second aim, which was to identify additional components of RNAi in budding yeast, is continued, with plans to identify the causative mutations in S. castellii strains with crippled RNAi. Identifying new components and modifiers would be important not only for understanding the yeast pathway but could also shed light on RNAi pathways in plants and animals, including humans. The third aim, which was to determine the mechanism of RNAi in budding yeast, is now extended to include other mechanistic experiments in both yeast and zebrafish. A more thorough understanding of this pathway in yeast and fish should provide additional insights regarding RNAi and related gene-silencing pathways in other eukaryotes and thereby contribute to fundamental knowledge relevant to human health. In an additional extension ofthe original proposal, the intracellular binding targets of mammalian proteins with dsRNA-binding domains will be identified and studied, with the intent of uncovering additional functions of dsRNA unrelated to RNAi. RELEVANCE (See instructions): The phenomenon known as RNA interference, fights viruses and genomic parasites. This proposed research studies the biochemical mechanism and biological functions of RNAi in zebrafish and yeast species closely related to baker's yeast. Achieving these goals will help researchers design new tools for studying the function of genes in various animal and fungal species (including some that cause diseases) and wil! provide insights into how cells normally determine the amount of protein that is oroduned from each aene